CA2738969A1 - Porphobilinogen deaminase gene therapy - Google Patents

Abstract

The present invention relates to nucleotide sequences coding for human porphobilinogen deaminase that are optimised for higher expression in mammalian cells. The invention further relates to DNA constructs comprising such optimised synthetic coding sequences for use in gene therapy of conditions caused by a deficiency in porphobilinogen deaminase, such as acute intermittent porphyria. Accordingly, the present invention relates to a nucleic acid or a nucleic acid construct comprising a nucleotide sequence coding for a human porphobilinogen deaminase, wherein at least 320 of the codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID NO: 1 or wherein at least 305 of the codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID NO: 3.

Description

Agent Ref: 77629/00002 1 Porphobilinogen Deaminase Gene Therapy 3 Field of the invention 4 The present invention relates to nucleotide sequences coding for human porphobilinogen deaminase and to nucleic acid constructs harbouring those sequences. The present invention 6 further relates to novel gene therapy vectors and methods for their use in treating and preventing 7 conditions caused by deficiency of porphobilinogen deaminase. More specifically, the gene 8 therapy vectors of the invention may be used in methods of alleviating the symptoms of such 9 conditions, including acute intermittent porphyria.
11 Background of the invention 12 Acute intermittent porphyria (AIP) is an inherited metabolic disease characterized by a 13 deficiency of porphobilinogen deaminase (PBGD), the third enzyme of the heme synthesis 14 pathway. The enzyme activity is -50% of normal in those who inherit the genetic trait. The disease is inherited in an autosomal dominant manner and is the most common of acute 16 porphyrias. Although it occurs in all races it is most prevalent in North Europe, mainly in 17 Sweden, Britain and Ireland. In USA and other countries the estimated prevalence is 5/100,000 18 and in Northern Sweden it is as high as 60-100/100,000. More than 225 mutations in the PBGD
19 gene have been described to date. The dominant clinical feature is an acute intermittent attack due to dysfunction of the nervous system, including abdominal pain and neurovisceral and 21 circulatory disturbances. Abdominal pain has been reported in 85-95% of cases and is the most 22 common feature, followed by or associated with the neurological changes.
Progression to 23 respiratory and bulbar paralysis and death may occur if AIP is not recognized and harmful drugs 24 are not withdrawn, such as drugs metabolized by the hepatic cytochrome P450 enzymes which may precipitate an attack. Sudden death may also occur as result of cardiac arrhythmia. Primary 26 liver cancer and impaired renal function sometimes occur as well.

27 An inherited deficiency of PBGD is not enough for the symptoms to appear. A
high 28 proportion of subjects that inherit PBGD mutation never develop porphyric symptoms, i.e. there 29 is very low clinical penetrance. Clinical symptoms in AIP carriers are associated with increased production and excretion of the porphyrin precursors delta-aminolevulinic acid (ALA) and 31 porphobilinogen (PBG) as result of increased demand of heme synthesis due to a drug or other 22094617.1 1 Agent Ref: 77629/00002 1 precipitating factors that provoke the acute attack. In these conditions PBGD deficiency limits 2 heme synthesis and as a result heme-mediated repression of ALA synthetase (ALAS 1) is 3 impaired. There is evidence indicating that the liver is the main source of the excess of porphyrin 4 precursors. These compounds remain elevated between attacks in those subjects prone to repeated porphyric crises and increase further during the crisis. They may decrease to normal if 6 the disease remains inactive for a long period of time.

7 Acute attacks usually occur after puberty and can be induced in latent individuals by 8 endocrine factors and steroid hormones and a variety of environmental factors including drug, 9 nutritional factors, restricted carbohydrate and caloric intake, smoking, steroid hormones and oral contraceptives, lead poisoning, intercurrent infections, surgery and psychological stress.
11 Drugs are among the most important factors that precipitate acute attacks and a list of safe drugs 12 is available in www.drugs-porphyria.com. Smoking, ethanol and drugs metabolized by CYP450, 13 greatly increase hepatic heme demand and result in the induction of ALAS 1, which increases the 14 production of porphryin precursors and precipitates an acute attack. Also, ALAS 1 is positively regulated by the peroxisome proliferator-activated receptor 7 coactivator 1 a (PGC 1 a), which is 16 induced in the liver during fasting. Among the precipitating factors steroid hormones seem to 17 play an important role. This concept is supported by the fact that the disease rarely manifests 18 before puberty and that oral contraceptives can exacerbate attacks in some females with PBGD
19 deficiency. Also women (80%) are affected more often than men (20%).

Acute attacks are treated with infusions of glucose and hemin (Normosang, Orphan 21 Europe). Glucose appears to antagonize the ALAS1 induction mediated by PGC-la. Hemin 22 restores the regulatory heme pool and suppresses hepatic ALAS I induction.
Some women 23 develop premenstrual attacks which can be prevented by gonadotropin-releasing hormone 24 (GnRH) analogs. Some patients exhibit recurrent acute attacks and significant, disabling neurological dysfunction. Advanced neurologic damage and subacute and chronic symptoms are 26 generally unresponsive to heme therapy. This is a life-threatening condition that can be cured 27 only by allogeneic liver transplantation that, in three patients to date, prevents the accumulation 28 of neurotoxic ALA and PBG. Nevertheless liver transplantation has limited availability of 29 compatible donors, and a significant morbidity and mortality.

Thus, gene-replacement therapy is a potential alternative to liver transplantation in these 31 patients where the liver function is entirely normal except for the PBGD
deficiency. Gene 22094617.1 2 Agent Ref: 77629/00002 1 therapy is a procedure consisting of the introduction of a specific gene into cells to control 2 disease through the use of vectors. The feasibility of gene delivery therapies aiming to correct the 3 hepatic enzyme defect are being explored in experimental models of AIP (AIP
mice). Adenoviral 4 vector-mediated gene transfer of PBGD to porphyric mice revealed short-term therapeutically efficacy as a result of the transient hepatic expression of PBGD (Johansson, 2004, Mol. Ther.
6 10(2):337-43). These results established a proof-of-principle, demonstrating that viral vector-7 mediated PBGD gene delivery may transiently ameliorate the severe manifestations of 8 phenobarbital-induced porphyric attacks in AIP mice.
9 EP 1 049 487 discloses the construction of rAAV vectors containing a human PBGD
cDNA only at a conceptual level.

11 There is however still a need in the art for improved vectors and protocols for AAV-12 mediated delivery of hPBGD to subjects in need thereof.

14 Summary of the invention According to the invention, there is provided a nucleotide sequence, i.e. a nucleic acid or 16 polynucleotide, coding for a human porphobilinogen deaminase (PBGD), wherein at least about 17 320 of the codons coding for the human porphobilinogen deaminase are identical to the codons 18 in SEQ ID NO: 1 or wherein at least about 305 of the codons coding for the human 19 porphobilinogen deaminase are identical to the codons in SEQ ID NO: 3.
Preferably, the nucleotide sequence coding for the human porphobilinogen deaminase has at least 95% identity 21 over the entire length of SEQ ID NO: 1 or 3, as determined by a Needleman and Wunsch global 22 alignment algorithm.
23 The "codons" in SEQ ID NOs: 1 and 3 refer to the codons in the frame beginning with 24 nucleotide 1 of SEQ ID NOs: 1 and 3, i.e. not the frame beginning with nucleotide 2 or 3 of SEQ
ID NOs: 1 and 3. That is to say, the first codon of SEQ NOs: 1 and 3 is indicated by nucleotide 26 numbers 1 to 3.

27 In another aspect, the invention relates to a nucleic acid construct comprising a nucleotide 28 sequence coding for a human porphobilinogen deaminase, wherein at least about 320 of the 29 codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID
NO: 1 or wherein at least about 305 of the codons coding for the human porphobilinogen 31 deaminase are identical to the codons in SEQ ID NO: 3. Preferably, in the nucleic acid construct 22094617.1 3 Agent Ref: 77629/00002 1 the nucleotide sequence coding for the human porphobilinogen deaminase is operably linked to a 2 promoter for expression in human cells, preferably a liver-specific promoter.
3 In a further aspect the invention relates to viral gene therapy vector comprising the 4 nucleotide sequence coding for the human porphobilinogen deaminase operably linked to a promoter for expression in human cells. Preferably, the vector is a recombinant parvoviral or 6 adeno-associated viral (AAV) vector.

7 In a further aspect, the invention pertains to a nucleic acid, nucleic acid construct or 8 parvoviral virion comprising the recombinant parvoviral or AAV vector that comprises the 9 nucleotide sequence coding for the human porphobilinogen deaminase.

In yet a further aspect, the invention relates to a pharmaceutical composition comprising 11 such a nucleic acid, nucleic acid construct or parvoviral virion and a pharmaceutically acceptable 12 carrier.

13 Further aspects of the invention relate to these nucleic acids, nucleic acid constructs or 14 parvoviral virions for use as a medicament, and for use in the treatment of a condition caused by a deficiency in porphobilinogen deaminase, wherein, preferably the condition is acute 16 intermittent porphyria (AIP).

17 The invention also relates to use of a nucleic acid, nucleic acid construct or parvoviral 18 virion of the invention for use in the manufacture of a medicament for use in the treatment of a 19 condition caused by a deficiency in porphobilinogen deaminase.
Also provided is a method for the delivery of a nucleotide sequence encoding 21 porphobilinogen deaminase to a mammal which method comprises:
22 a. providing a nucleic acid, nucleic acid construct or parvoviral virion of the invention; and 23 b. administering said nucleic acid, nucleic acid construct or parvoviral virion to a mammal under 24 conditions that result in the expression of protein at a level that provides a therapeutic effect in said mammal.

26 The invention also relates to a method for treating a condition caused by a deficiency in 27 porphobilinogen deaminase wherein the method comprises the step of administering an effective 28 amount of a pharmaceutical composition comprising a nucleic acid, nucleic acid construct or 29 parvoviral virion with the nucleotide sequence coding for the human porphobilinogen deaminase, to a subject with a porphobilinogen deaminase deficiency, wherein, preferably the condition is 31 acute intermittent porphyria.

22094617.1 4 Agent Ref: 77629/00002 2 Description of the invention 3 Definitions 4 A "nucleic acid" includes any molecule composed of or comprising monomeric nucleotides. The term "nucleotide sequence" may be used interchangeably with "nucleic acid"
6 herein. A nucleic acid may be an oligonucleotide or a polynucleotide. A
nucleic acid may be a 7 DNA or an RNA. A nucleic acid may be chemically modified or artificial.
Artificial nucleic 8 acids include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as 9 glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
Also, 11 phosphorothioate nucleotides may be used. Other deoxynucleotide analogs include 12 methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and 13 oligoribonucleotide phosphorothioates and their 2'-0-allyl analogs and 2'-O-methylribonucleotide 14 methylphosphonates which may be used in a nucleic acid of the invention.

A "nucleic acid construct" is herein understood to mean a man-made nucleic acid molecule 16 resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic 17 acid molecule, either single- or double-stranded, which has been modified to contain segments of 18 nucleic acids, which are combined and juxtaposed in a manner, which would not otherwise exist 19 in nature. A nucleic acid construct usually is a "vector", i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.
21 One type of nucleic acid construct is an "expression cassette" or "expression vector".
22 These terms refers to nucleotide sequences that are capable of effecting expression of a gene in 23 host cells or host organisms compatible with such sequences. Expression cassettes or expression 24 vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression 26 may also be present, such as expression enhancer elements.

27 The term "homologous" when used to indicate the relation between a given (recombinant) 28 nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to 29 mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species. The term "heterologous" may be used to indicate that in nature 22094617.1 5 Agent Ref: 77629/00002 1 the nucleic acid or polypeptide molecule is produced by a host cell or organisms of a different 2 species.
3 As used herein, the term "operably linked" refers to a linkage of polynucleotide (or 4 polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a 6 transcription regulatory sequence is operably linked to a coding sequence if it affects the 7 transcription of the coding sequence. Operably linked means that the DNA
sequences being 8 linked are typically contiguous and, where necessary to join two protein encoding regions, 9 contiguous and in reading frame.

"Expression control sequence" refers to a nucleic acid sequence that regulates the 11 expression of a nucleotide sequence to which it is operably linked. An expression control 12 sequence is "operably linked" to a nucleotide sequence when the expression control sequence 13 controls and regulates the transcription and/or the translation of the nucleotide sequence. Thus, 14 an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing 16 signals for introns, and stop codons. The term "expression control sequence" is intended to 17 include, at a minimum, a sequence whose presence are designed to influence expression, and can 18 also include additional advantageous components. For example, leader sequences and fusion 19 partner sequences are expression control sequences. The term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are 21 removed from the sequence. It can also include the design of the nucleic acid sequence such that 22 undesirable potential splice sites are removed. It includes sequences or polyadenylation 23 sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 24 3'-end of a mRNA, which may be referred to as polyA sequences. It also can be designed to enhance mRNA stability. Expression control sequences which affect the transcription and 26 translation stability, e.g., promoters, as well as sequences which effect the translation, e.g., 27 Kozak sequences, suitable for use in insect cells are well known to those skilled in the art.
28 Expression control sequences can be of such nature as to modulate the nucleotide sequence to 29 which it is operably linked such that lower expression levels or higher expression levels are achieved.

22094617.1 6 Agent Ref: 77629/00002 1 As used herein, the term "promoter" or "transcription regulatory sequence"
refers to a 2 nucleic acid fragment that functions to control the transcription of one or more coding sequences, 3 and is located upstream with respect to the direction of transcription of the transcription initiation 4 site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA
sequences, 6 including, but not limited to transcription factor binding sites, repressor and activator protein 7 binding sites, and any other sequences of nucleotides known to one of skill in the art to act 8 directly or indirectly to regulate the amount of transcription from the promoter, including e.g.
9 attenuators or enhancers, but also silencers. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter 11 is a promoter that is physiologically or developmentally regulated, e.g. by the application of a 12 chemical inducer. A "tissue specific" promoter is only active in specific types of tissues or cells.
13 A "3' UTR" or "3' non-translated sequence" (also often referred to as 3' untranslated 14 region, or 3'end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all 16 eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof). After 17 termination of transcription, the mRNA transcript may be cleaved downstream of the 18 polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the 19 mRNA to the cytoplasm (where translation takes place).
The terms "substantially identical", "substantial identity" or "essentially similar" or 21 "essential similarity" means that two peptide or two nucleotide sequences, when optimally 22 aligned, such as by the programs GAP or BESTFIT using default parameters, share at least a 23 certain percentage of sequence identity as defined elsewhere herein. GAP
uses the Needleman 24 and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP
26 default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap 27 extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used 28 is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 29 1992, PNAS 89, 915-919). It is clear than when RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA
31 sequence is considered equal to uracil (U) in the RNA sequence. Sequence alignments and scores 22094617.1 7 Agent Ref: 77629/00002 1 for percentage sequence identity may be determined using computer programs, such as the GCG
2 Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, 4 USA or the open-source software Emboss for Windows (current version 2.7.1-07). Alternatively percent similarity or identity may be determined by searching against databases such as FASTA, 6 BLAST, etc.
7 In this document and in its claims, the verb "to comprise" and its conjugations are used in 8 its non-limiting sense to mean that items following the word are included, but items not 9 specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, 11 unless the context clearly requires that there be one and only one of the elements. The indefinite 12 article "a" or "an" thus usually means "at least one".

14 Detailed description of the invention The invention pertains to a nucleotide sequences coding for porphobilinogen deaminase.
16 The nucleotide sequence coding for porphobilinogen deaminase is preferably a synthetic 17 nucleotide sequence. The term "synthetic nucleotide sequence" is herein understood to mean that 18 the nucleotide sequence does not occur as such in nature, but rather was designed, engineered 19 and/or constructed by human intervention. The term "synthetic" thus does not necessarily imply that the sequence is exclusively and/or entirely obtained through chemical synthesis. Rather, 21 although parts of the synthetic sequence may at one stage have been obtained through chemical 22 synthesis, molecules comprising a synthetic sequence of the invention will usually be obtained 23 from biological sources such as (cultured, for example recombinant) cells.
24 The nucleotide sequence of the invention may encode an erythroid or a non-erythroid porphobilinogen deaminase. Preferably, the nucleotide sequence encodes a porphobilinogen 26 deaminase of human origin. The nucleotide sequence may thus encode any naturally occurring 27 amino acid sequence of an allelic form of a human porphobilinogen deaminase. However, 28 explicitly included in the inventions are nucleotide sequence that encode engineered muteins of 29 porphobilinogen deaminases having one more amino acid substitutions, deletions and/or insertions compared to e.g. a naturally occurring human amino acid sequence.
Preferably the 31 nucleotide sequence encodes a protein that has porphobilinogen deaminase activity (EC 2.5.1.61) 22094617.1 8 Agent Ref: 77629/00002 1 as may be determined by an assay as e.g. described by Wright and Lim (1983, Biochem. J. 213:
2 85-88).
3 In a preferred embodiment of the invention, the nucleotide sequence coding for a 4 porphobilinogen deaminase has an improved codon usage bias for the human cell as compared to naturally occurring nucleotide sequence coding for the deaminases. The adaptiveness of a 6 nucleotide sequence encoding a porphobilinogen deaminase to the codon usage of human cells 7 may be expressed as codon adaptation index (CAI). A codon adaptation index is herein defined 8 as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon 9 usage of highly expressed human genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI
11 is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons 12 and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, 13 with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li 14 , 1987, Nucleic Acids Research 15: 1281-1295; also see: Kim et al., Gene.
1997, 199:293-301;
zur Megede et al., Journal of Virology, 2000, 74: 2628-2635). Preferably, a nucleotide sequence 16 encoding a porphobilinogen deaminase has a CAI of at least 0.8, 0.85, 0.90, 0.92, 0.94, 0.95, 17 0.96 or 0.97.

18 In a preferred nucleotide sequence of the invention, at least 320, 330, 340, 345, 350, 355, 19 356, 357, 358, 359, 360, or 361 of all codons coding for the non-erythroid porphobilinogen deaminase are identical to the codons (in corresponding positions) in SEQ ID
NO: 1. More 21 preferably the nucleotide sequence codes for the amino acid sequence of SEQ
ID NO: 2.
22 Alternatively, in a preferred nucleotide sequence of the invention, at least 305, 310, 315, 23 320, 325, 330, 335, 340, 341, 342, 343, or 344 of the codons coding for the erythroid 24 porphobilinogen deaminase are identical to the codons (in corresponding positions) in SEQ ID
NO: 3. More preferably the nucleotide sequence codes for the amino acid sequence of SEQ ID
26 NO: 4.
27 The "codons" in SEQ ID NOs: 1 and 3 refer to the codons in the frame beginning with 28 nucleotide 1 of SEQ ID NOs: 1 and 3, i.e. not the frame beginning with nucleotide 2 or 3 of SEQ
29 ID NOs: 1 and 3. That is to say, the first codon of SEQ NOs: 1 and 3 is indicated by nucleotide numbers 1 to 3.

22094617.1 9 Agent Ref: 77629/00002 1 Another preferred nucleotide sequence of the invention codes for a polypeptide with 2 porphobilinogen deaminase activity, whereby the nucleotide sequence has at least 95, 96, 97, 98 3 or 99% nucleotide sequence identity over its entire length with SEQ ID NO: 1 or 3, as 4 determined by a Needleman and Wunsch global alignment algorithm. More preferably the nucleotide sequence codes for the amino acid sequences of SEQ ID NO: 2 or 4.
6 In a particularly preferred embodiment of invention, the nucleotide sequence has the 7 nucleotide sequence of SEQ ID NO: 1 or 3.
8 In a further aspect the invention pertains to a nucleic acid construct comprising a 9 nucleotide sequence of the invention as herein defined above. In the nucleic acid construct the nucleotide sequence encoding the porphobilinogen deaminase preferably is operably linked to a 11 mammalian cell-compatible expression control sequence, e.g., a promoter.
Many such promoters 12 are known in the art (see Sambrook and Russell, 2001, supra). Constitutive promoters that are 13 broadly expressed in many cell types, such as the CMV promoter may be used.
However, 14 promoters that are inducible, tissue-specific, cell-type-specific, or cell cycle-specific may be preferred. In a preferred embodiment, the nucleotide sequence encoding the porphobilinogen 16 deaminase is operably linked to a liver-specific promoter. Liver-specific promoters are 17 particularly preferred for use in conjunction the non-erythroid deaminase.
Preferably, in a 18 construct of the invention an expression control sequence for liver-specific expression are e.g.
19 selected from the group consisting of an al-anti-trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a thyroxin-binding globulin (TBG) promoter, 21 an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an 22 AAT promoter combined with the mouse albumin gene enhancer (Ealb) element and an 23 apolipoprotein E promoter. Other examples include the E2F promoter for tumour-selective, and, 24 in particular, neurological cell tumour-selective expression (Parr et al., 1997, Nat. Med. 3:1145-9) or the IL-2 promoter for use in mononuclear blood cells (Hagenbaugh et al., 1997, J Exp Med;
26 185: 2101-10). In a particularly preferred embodiment of the invention, wherein the promoter 27 has the sequence of SEQ ID NO: 5.

28 In a further preferred embodiment of the nucleic acid construct of the invention a 3'UTR
29 (or 3' non-translated sequence) may be located downstream of the nucleotide sequence encoding the porphobilinogen deaminase. Suitable 3'UTR sequences are available to the skilled person.
31 They may be derived from any mammalian and preferably human gene and will usually 22094617.1 10 Agent Ref: 77629/00002 1 comprise a transcription termination site and a polyadenylation signal (such as e.g. AAUAAA or 2 variants thereof). In a particularly preferred embodiment the nucleic acid construct comprises a 3 3'UTR derived from the human PBGD gene such as e.g. SEQ ID NO: 6.

4 In another preferred embodiment of the nucleic acid construct of the invention, the expression control sequence that is operably linked to the nucleotide sequence encoding the 6 porphobilinogen deaminase, is preceded upstream by a polyA insulator to terminate run-through 7 transcription from possible upstream transcription units. A 3'UTR as described above and 8 preferably at least comprising a transcription termination sequence may be used for this purpose.
9 A preferred polyA insulator is a synthetic polyA insulator having the sequence of SEQ ID NO: 7.
In one preferred embodiment the nucleic acid construct, the invention may comprise a 11 Kozak consensus sequence around the initiation codon of the nucleotide sequence encoding the 12 porphobilinogen deaminase. The Kozak consensus sequence is herein defined as 13 GCCRCC(AUG)A (SEQ ID NO: 8), wherein R is a purine (i.e. A, adenosine or G, guanosine) 14 and wherein (AUG) stands for the initiation codon of the porphobilinogen deaminase coding sequence. Although in the usual Kozak consensus sequence the nucleotide directly following the 16 AUG initiation codon is a G (guanosine), in context of the present invention this nucleotide 17 preferably is an A (adenosine) in both the erythroid and non- erythroid porphobilinogen 18 deaminase coding sequence. In a preferred embodiment the Kozak consensus sequence may be 19 preceded by another GCC triplet.

In an additional aspect the invention relates to a nucleic acid construct comprising a 21 nucleotide sequence encoding the porphobilinogen deaminase that is operably linked to an 22 expression control sequence as defined herein above, wherein the construct is an expression 23 vector that is suitable for gene therapy of mammals, preferably gene therapy of humans. A
24 preferred nucleic acid construct according to the invention is a viral gene therapy vector. Viral gene therapy vectors are well known in the art and e.g. include vectors based on an adenovirus, 26 and members of the Parvoviridae family, such as an adeno-associated virus (AAV), or a herpes 27 virus, pox virus or retrovirus. A preferred viral gene therapy vector is an AAV, adenoviral or a 28 lentiviral vector.

29 Particularly preferred gene therapy vectors in the context of the present invention are parvoviral vectors. Thus, in this preferred aspect the invention relates the use of animal 31 parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and the 22094617.1 11 Agent Ref: 77629/00002 1 components thereof (e.g., an animal parvovirus genome) for use as vectors for introduction 2 and/or expression of the nucleotide sequences encoding a porphobilinogen deaminase in 3 mammalian cells.
4 Viruses of the Parvoviridae family are small DNA animal viruses. The family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the 6 Densovirinae, which infect insects. Members of the subfamily Parvovirinae are herein referred to 7 as the parvoviruses and include the genus Dependovirus. As may be deduced from the name of 8 their genus, members of the Dependovirus are unique in that they usually require co-infection 9 with a helper virus such as adenovirus or herpes virus for productive infection in cell culture.
The genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 1, 2, 11 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other 12 warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses).
13 Further information on parvoviruses and other members of the Parvoviridae is described in 14 Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication,"
Chapter 69 in Fields Virology (3d Ed. 1996). For convenience the present invention is further exemplified and 16 described herein by reference to AAV. It is however understood that the invention is not limited 17 to AAV but may equally be applied to other parvoviruses.
18 The genomic organization of all known AAV serotypes is very similar. The genome of 19 AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the 21 non-structural replication (Rep) proteins and the structural (VP) proteins.
The VP proteins (VP1, 22 -2 and -3) form the capsid. The terminal 145 nt are self-complementary and are organized so that 23 an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These 24 hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild-type (wt) AAV infection in mammalian cells 26 the Rep genes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, 27 respectively and both Rep proteins have a function in the replication of the viral genome. A
28 splicing event in the Rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, 29 Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also 31 in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.

22094617.1 12 Agent Ref: 77629/00002 1 A "recombinant parvoviral or AAV vector" (or "rAAV vector") herein refers to a vector 2 comprising one or more polynucleotide sequences of interest, genes of interest or "transgenes"
3 that are flanked by at least one parvoviral or AAV inverted terminal repeat sequences (ITRs).
4 Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products (i.e. AAV
Rep and Cap 6 proteins). When an rAAV vector is incorporated into a larger nucleic acid construct (e.g. in a 7 chromosome or in another vector such as a plasmid or baculovirus used for cloning or 8 transfection), then the rAAV vector is typically referred to as a "pro-vector" which can be 9 "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions. Thus, in a further aspect the invention relates to a nucleic acid 11 construct comprising a nucleotide sequence encoding a porphobilinogen deaminase as herein 12 defined above, wherein the nucleic acid construct is a recombinant parvoviral or AAV vector and 13 thus comprises at least one parvoviral or AAV ITR. Preferably, in the nucleic acid construct the 14 nucleotide sequence encoding the porphobilinogen deaminase is flanked by parvoviral or AAV
ITRs on either side. Any parvoviral or AAV ITR may be used in the constructs of the invention, 16 including ITRs from AAV 1, AAV2, AAV4, AAV5, AAV6, AAV8 and/or AAV9. ITRs of 17 AAV2 are most preferred. Examples of preferred ITR sequences for use in preferred nucleic acid 18 constructs of the invention are given SEQ ID NO: 9 (left or upstream ITR) and SEQ ID NO: 10 19 (right or downstream ITR).
AAV is able to infect a number of mammalian cells. See, e.g., Tratschin et al.
(1985, Mol.
21 Cell Biol. 5:3251-3260) and Grimm et al. (1999, Hum. Gene Ther. 10:2445-2450). However, 22 AAV transduction of human synovial fibroblasts is significantly more efficient than in similar 23 murine cells, Jennings et al., Arthritis Res, 3:1 (2001), and the cellular tropicity of AAV differs 24 among serotypes. See, e.g., Davidson et al. (2000, Proc. Natl. Acad. Sci.
USA, 97:3428-3432), who discuss differences among AAV2, AAV4, and AAV5 with respect to mammalian CNS cell 26 tropism and transduction efficiency and see Goncalves, 2005, Virol J.
2(1):43 who discusses 27 approaches to modification of AAV tropism. For transduction of liver cells rAAV virions with 28 AAV1, AAV8 and AAV5 capsid proteins are preferred (Nathwani et al., 2007, Blood 109(4):
29 1414-1421; Kitajima et al., 2006, Atherosclerosis 186(1):65-73), of which is rAAV virions with AAV5 capsid proteins may be most preferred.

22094617.1 13 Agent Ref: 77629/00002 1 AAV sequences that may be used in the present invention for the production of rAAV
2 vectors in insect cells can be derived from the genome of any AAV serotype.
Generally, the 3 AAV serotypes have genomic sequences of significant homology at the amino acid and the 4 nucleic acid levels. This provides an identical set of genetic functions to produce virions which are essentially physically and functionally equivalent. For the genomic sequence of the various 6 AAV serotypes and an overview of the genomic similarities see e.g. GenBank Accession number 7 U89790; GenBank Accession number J01901; GenBank Accession number AF043303;
8 GenBank Accession number AF085716; Chlorini et al. (1997, J. Vir. 71: 6823-33); Srivastava et 9 al. (1983, J. Vir. 45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319);
Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74: 8635-47). rAAV serotypes 1, 2, 3, 4 and 5 11 are preferred source of AAV nucleotide sequences for use in the context of the present invention.
12 Preferably the AAV ITR sequences for use in the context of the present invention are derived 13 from AAVI, AAV2, and/or AAV4. Likewise, the Rep (Rep78/68 and Rep52/40) coding 14 sequences are preferably derived from AAV1, AAV2, and/or AAV4. The sequences coding for the viral proteins (VP) VPl, VP2, and VP3 capsid proteins for use in the context of the present 16 invention may however be taken from any of the known 42 serotypes, more preferably from 17 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed 18 AAV-like particles obtained by e.g. capsid shuffling techniques and AAV
capsid libraries.

19 AAV Rep and ITR sequences are particularly conserved among most serotypes.
The Rep78 proteins of various AAV serotypes are e.g. more than 89% identical and the total nucleotide 21 sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is around 22 82% (Bantel-Schaal et al., 1999, J. Virol., 73(2):939-947). Moreover, the Rep sequences and 23 ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally 24 substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells. US2003148506 reports that AAV Rep and ITR sequences also efficiently 26 cross-complement other AAV Rep and ITR sequences in insect cells.
27 The AAV VP proteins are known to determine the cellular tropicity of the AAV virion.
28 The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes 29 among different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV
31 particles comprising the capsid proteins of one serotype (e.g., AAV5) and the Rep and/or ITR
22094617.1 14 Agent Ref: 77629/00002 1 sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV
particles are a part 2 of the present invention. Herein, a pseudotyped rAAV particle may be referred to as being of the 3 type "x/y", where "x" indicates the source of ITRs and "y" indicates the serotype of capsid, for 4 example a 2/5 rAAV particle has ITRs from AAV2 and a capsid from AAV5.
Modified "AAV" sequences also can be used in the context of the present invention, e.g.
6 for the production of rAAV vectors in insect cells. Such modified sequences e.g. include 7 sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, 8 at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity 9 (e.g., a sequence having from about 75% to about 99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP can be 11 used in place of wild-type AAV ITR, Rep, or VP sequences.
12 Although similar to other AAV serotypes in many respects, AAV5 differs from other 13 human and simian AAV serotypes more than other known human and simian serotypes. In view 14 thereof, the production of rAAV5 can differ from production of other serotypes in insect cells.
Where methods of the invention are employed to produce rAAV5, it is preferred that one or 16 more constructs comprising, collectively in the case of more than one construct, a nucleotide 17 sequence comprising an AAV5 ITR, a nucleotide sequence comprises an AAV5 Rep coding 18 sequence (i.e. a nucleotide sequence comprises an AAV5 Rep78). Such ITR and Rep sequences 19 can be modified as desired to obtain efficient production of rAAV5 or pseudotyped rAAV5 vectors in insect cells. E.g., the start codon of the Rep sequences can be modified, VP splice sites 21 can be modified or eliminated, and/or the VP1 start codon and nearby nucleotides can be 22 modified to improve the production of rAAV5 vectors in the insect cell.
23 Preferred adenoviral vectors are modified to reduce the host response as reviewed by 24 Russell (2000, J. Gen. Virol. 81: 2573-2604), or as described in US20080008690 and by Zaldumbide and Hoeben (Gene Therapy 2008:239-246).
26 The invention thus also relates to a parvoviral virion comprising a nucleic acid construct as 27 herein defined above, and parvoviral capsid protein as defined herein above.

28 In an additional aspect, the invention relates to a method for producing a recombinant 29 parvoviral (for example rAAV) virion (comprising a recombinant parvoviral (rAAV) vector as defined above) in an insect cell. Preferably, the method comprises the steps of. (a) culturing an 31 insect cell as defined herein under conditions such that recombinant parvoviral (for example 22094617.1 15 Agent Ref: 77629/00002 1 rAAV) vector is produced; and, (b) recovery of the recombinant parvoviral (for example rAAV) 2 vector. It is understood here that the recombinant parvoviral (rAAV) vector produced in the 3 method preferably is an infectious parvoviral or AAV virion that comprise the recombinant 4 parvoviral (rAAV) vector nucleic acids. Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art and 6 described e.g. in the above cited references on molecular engineering of insects cells. Preferred 7 methods and constructs for the production of rAAV virions of the invention are disclosed in e.g.
8 W02007/046703 and W02007/148971.
9 Preferably the method for producing recombinant parvoviral virions further comprises the step of affinity-purification of the (virions comprising the) recombinant parvoviral (rAAV) 11 vector using an anti-AAV antibody, preferably an immobilised antibody. The anti-AAV antibody 12 preferably is an monoclonal antibody. A particularly suitable antibody is a single chain camelid 13 antibody or a fragment thereof as e.g. obtainable from camels or llamas (see e.g. Muyldermans, 14 2001, Biotechnol. 74: 277-302). The antibody for affinity-purification of rAAV preferably is an antibody that specifically binds an epitope on a AAV capsid protein, whereby preferably the 16 epitope is an epitope that is present on capsid proteins of more than one AAV serotype. E.g. the 17 antibody may be raised or selected on the basis of specific binding to AAV2 capsid but at the 18 same time also it may also specifically bind to AAV1, AAV3 and AAV5 capsids.
19 Also, the invention pertains to a parvoviral virion as herein defined above, for use as a medicament. That is to say, the invention provides a parvoviral virion of the invention for use in 21 the method of treatment of the human or animal body by therapy.
22 The invention further pertains to a parvoviral virion as herein defined above, for use in the 23 treatment of a condition caused by a deficiency in porphobilinogen deaminase. Preferably such 24 condition is acute intermittent porphyria. A nucleic acid or a nucleic acid construct of the invention are also suitable for such use.
26 Accordingly, the invention relates to a nucleic acid, a nucleic acid construct or a parvoviral 27 virion of the invention for use in the preparation of medicament for use in a method of treatment 28 of a condition caused by a deficiency in porphobilinogen deaminase.
Preferably such condition is 29 acute intermittent porphyria. Such treatment may alleviate, ameliorate, reduce the severity of one or more symptoms of AIP, for example reducing the incidence or severity of an attack. For 31 example, treatment according to the invention may alleviate, ameliorate, reduce the severity of 22094617.1 16 Agent Ref: 77629/00002 1 dysfunction of the nervous system, abdominal pain or neurovisceral and/or circulatory 2 disturbances.
3 Further, the invention pertains to a pharmaceutical composition comprising a parvoviral 4 virion as herein defined above. The pharmaceutical composition further preferably comprises a pharmaceutically acceptable carrier. Any suitable pharmaceutically acceptable carrier or 6 excipient can be used in the present compositions (See e.g., Remington: The Science and 7 Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).
8 Preferred pharmaceutical forms would be in combination with sterile saline, dextrose solution, or 9 buffered solution, or other pharmaceutically acceptable sterile fluids.
Alternatively, a solid carrier, may be used such as, for example, microcarrier beads.
11 Pharmaceutical compositions are typically sterile and stable under the conditions of 12 manufacture and storage. Pharmaceutical compositions may be formulated as a solution, 13 microemulsion, liposome, or other ordered structure suitable to accommodate high drug 14 concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, 16 and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, 17 by the use of a coating such as lecithin, by the maintenance of the required particle size in the 18 case of dispersion and by the use of surfactants. In many cases, it will be preferable to include 19 isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by 21 including in the composition an agent which delays absorption, for example, monostearate salts 22 and gelatin. The parvoviral virion may be administered in a time or controlled release 23 formulation, for example in a composition which includes a slow release polymer or other 24 carriers that will protect the compound against rapid release, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may for example 26 be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, 27 polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG).
28 The invention also provides a method for the delivery of a nucleotide sequence encoding 29 porphobilinogen deaminase to a mammal which method comprises:
a. providing a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical 31 composition as defined herein; and 22094617.1 17 Agent Ref: 77629/00002 1 b. administering said nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical 2 composition to a mammal under conditions that result in the expression of protein at a level that 3 provides a therapeutic effect in said mammal.
4 Also, the invention relates to a method for treating a condition caused by a deficiency in porphobilinogen deaminase wherein the method comprises the step of administering an effective 6 amount of a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition 7 as defined herein above to a subject with a porphobilinogen deaminase deficiency. Preferably the 8 subject is suffering from the condition acute intermittent porphyria.
9 In the treatment or therapy according to the invention, a condition caused by a deficiency in porphobilinogen deaminase is treated by administering to a subject an effective amount of a 11 nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition as defined 12 herein.
13 The condition of a patient suffering from a such a condition can be improved by 14 administration of a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition of the invention. A therapeutically effective amount of a nucleic acid, nucleic acid 16 construct, parvoviral virion or pharmaceutical composition of the invention may be given to a 17 patient in need thereof.
18 The a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition 19 will typically be included in a pharmaceutical composition, optionally in combination with a pharmaceutical carrier, diluent and/or adjuvant. Such compositions include the nucleic acid, 21 nucleic acid construct, parvoviral virion or pharmaceutical composition in an effective amount, 22 sufficient to provide a desired therapeutic or prophylactic effect, and a pharmaceutically 23 acceptable carrier or excipient. An "effective amount" includes a therapeutically effective 24 amount or a prophylactically effective amount.
A "therapeutically effective amount" refers to an amount effective, at dosages and for 26 periods of time necessary, to achieve the desired therapeutic result, such as elevation of PBGD
27 activity. A therapeutically effective amount of a nucleic acid, nucleic acid construct, parvoviral 28 virion or pharmaceutical composition may vary according to factors such as the disease state, 29 age, sex, and weight of the individual, and the ability of the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition to elicit a desired response in the individual.
31 Dosage regimens may be adjusted to provide the optimum therapeutic response. A

22094617.1 18 Agent Ref: 77629/00002 1 therapeutically effective amount is also typically one in which any toxic or detrimental effects of 2 the nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical composition are 3 outweighed by the therapeutically beneficial effects.
4 A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or 6 inhibiting various conditions, including a condition associated with a reduction in PBGD levels.
7 A prophylactic dose may be used in subjects prior to or at an earlier stage of disease, and a 8 prophylactically effective amount may be more or less than a therapeutically effective amount in 9 some cases.
In particular embodiments, a range for therapeutically or prophylactically effective 11 amounts of a nucleic acid, nucleic acid construct, parvoviral virion or pharmaceutical 12 composition may be from 1 x 1012 and 1 x 1013 genome copy (gc) /kg, for example from l x 1011 to 13 1 x 1012 gc/kg. It is to be noted that dosage values may vary with the severity of the condition to 14 be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or 16 supervising the administration of the compositions. Dosage ranges set forth herein are exemplary 17 only and do not limit the dosage ranges that may be selected by medical practitioners.
18 For gene therapy vectors, such as the parvoviral virion of the present invention, the dosage 19 to be administered may depend to a large extent on the condition and size of the subject being treated as well as the therapeutic formulation, frequency of treatment and the route of 21 administration. Regimens for continuing therapy, including dose, formulation, and frequency 22 may be guided by the initial response and clinical judgment. The parenteral route of injection 23 into the interstitial space of tissue may be preferred, although other parenteral routes, such as 24 inhalation of an aerosol formulation, may be required in specific administration. In some protocols, a formulation comprising the gene and gene delivery system in an aqueous carrier is 26 injected into tissue in appropriate amounts.
27 The tissue target may be specific, for example the liver tissue, or it may be a combination 28 of several tissues, for example the muscle and liver tissues. Exemplary tissue targets may include 29 liver, skeletal muscle, heart muscle, adipose deposits, kidney, lung, vascular endothelium, epithelial and/or hematopoietic cells.
31 In one embodiment, the effective dose range for small animals (mice), following intramuscular 22094617.1 19 Agent Ref: 77629/00002 1 injection, maybe between 1 X, 0 12 and 1 x 10' 3 genome copy (gc) /kg, and for larger animals (cats) 2 and for human subjects, between 1 x 1011 and IX 10' 2 gc/kg.
3 The amount of active a nucleic acid, nucleic acid construct, parvoviral virion or 4 pharmaceutical composition in the compositions of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be 6 adjusted to provide the optimum therapeutic response. For example, a single bolus may be 7 administered, several divided doses may be administered over time or the dose may be 8 proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
9 It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to 11 physically discrete units suited as unitary dosages for subjects to be treated; each unit containing 12 a predetermined quantity of active compound calculated to produce the desired therapeutic effect 13 in association with the required pharmaceutical carrier. The specification for the dosage unit 14 forms of the invention may be dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and by the limitations inherent in the art of 16 compounding such an active compound for the treatment of a condition in individuals.
17 As used herein "pharmaceutically acceptable carrier" or "exipient" includes any and all 18 solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption 19 delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration, which includes intravenous, intraperitoneal or 21 intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral 22 administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or 23 dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions 24 or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the 26 active compound, use thereof in the pharmaceutical compositions of the invention is 27 contemplated.
28 Supplementary active compounds can also be incorporated into the pharmaceutical 29 compositions of the invention. Guidance on co-administration of additional therapeutics may for example be found in the Compendium of Pharmaceutical and Specialties (CPS) of the Canadian 31 Pharmacists Association.

22094617.1 20 Agent Ref: 77629/00002 2 Description of the figures 3 Figure 1. Dose effect of AAV2/8-hPBGD injection on heme precursor excretion in male 4 AIP mice. AIP mice have 25-30% of normal PBGD activity, resulting from a disruption in one allele of PBGD and a partial disruption in the other allele. LUC is luciferase reporter construct;
6 PBGD is various doses of rAAV-PBGD vector. Expressed as genome copies per kilogam; Pb is 7 phenobarbital.

9 Figure 2. Urinary excretion of PBG and ALA in female AIP mice after phenobarbital-induced acute attacks after the administration of 5 x 1012 vg/kg of therapeutic vector ssAAV2/8-11 hPBGD or control vector ssAAV2/8-Luc. LUC is luciferase reporter construct;
PBGD is various 12 dosages of rAAV-PBGD constructs; Pb is phenobarbital.

14 Figure 3. PBGD transgene expression in the liver of male AIP mice as measured by Western blot analysis.

17 Figure 4. PBGD transgene expression in the liver of female AIP mice as measured by 18 Western blot analysis.

Figure 5. Hepatic PBGD activity in AIP male mice transduced with ssAAV2/8 at different 21 doses, 3 months post-injection.

23 Figure 6. PBGD activity in the liver of AIP female mice transduced with ssAAV2/8 vector 24 carrying the luciferase or the PBGD gene, 3 months post-injection.
26 Figure 7. Comparison of in vivo enzymatic activity of PBGD in wild type (WT
mice) and 27 AIP mice (AIP mice) upon hydrodynamic injection of plasmid DNA constructs comprising the 28 wild type PBGD coding sequence (PBGD) and the synthetic, i.e. codon-optimised, PBGD coding 29 sequence of SEQ ID NO: 1 (coPBGD). The levels of vector DNA present in the liver was confirmed by using primers that hybridise to the transgene. Q-PCR-based ratios between the 22094617.1 21 Agent Ref: 77629/00002 1 DNA copies of the endogenous housekeeping gene GADPH and the PBGD transgene are 2 indicated above the PBGD enzymatic activities and do not show a significant difference.

4 Figures 8 and 9. AAV2/5-PBGD protect male (Figure 8) and female (Figure 9) mice against phenobarbital-induced acute porphyric attacks. Basal levels of ALA and PBG in AIP
6 mice as well as ALA and PBG level after first, second and third phenobarbital-induced acute 7 porphyric attacks are shown in mice treated with a dose of 5e12 gc/kg control vector (AAV2/5-8 EalbAAT-Luciferase) and mice treated with 5e12 gc/kg AAV8- and AAV5-PBGD
vectors.

Figure 10. PBGD enzymatic activity in liver homogenates after AAV8-PBGD and 11 LUC administration (n=4-6).

13 Fij~ure A. PBGD enzyme activity in liver homogenates of AIP male mice 1, 2 or 3 14 months after a single injection of 1.25x1011 gc of ssAAV2/5 vector carrying the PBGD cDNA
from human (wt PBGD) or codon optimised PBGD (coPBGD; SEQ ID NO: 1). B. Semi-16 quantitative PCR analysis of vector copy levels at 1, 2 and 3 months post-injection. C.
17 Representative immunohistochemical analysis of livers from male animals injected with an 18 ssAAV2/5 vector carrying the wtPBGD or coPBGD transgene under the control of the liver 19 specific promoter. D. Proportion of cells stained with PBGD antibody from each cohort of animals.

22 Figure 12. Neurological evaluation of peripheral neuropathy in AIP mice.
The percentage 23 of large axons (5 m in diameter) and axon density was performed in AIP mice of both genders 24 that received three different doses of rAAV2/8-PBGD. Untreated and wild type (WT) act as controls.

27 Figure 13. Rotarod analysis of AIP mouse motor coordination and muscular performance.
28 The length in time that male and female AIP mice could stay on a rotating dowel was measured 29 at the start of the study and upon induction of a porphyria attack 90 days after administration of AAV2/8-PBGD. The porphyric attack was induced by intraperitoneal injection of increasing 31 doses of phenobarbital every 24 hours for four days.

22094617.1 22 Agent Ref: 77629/00002 1 Examples 2 Example 1: AAV-mediated liver-specific expression of porphobilinogen deaminase reverts 3 biochemical alterations and protects against motor neuropathy in a mouse model of acute 4 intermittent porphyria 1.1 Materials and Methods.
6 1.1.1 Animal model 7 The acute intermittent porphyria (AIP) mice were generated by gene targeting as described 8 by Lindberg et al. (Nature genetics, 1996). The Ti and T2 transgenic strains were kindly 9 provided by Prof. Urs Meyer from the University of Basel and the animal facility of the University of Navarra has established a colony of these animals. In Ti transgenic animals a 11 Neomycin gene has been inserted in the first exon of the PBGD gene and homozygous animal 12 have a 45% loss of the PBGD activity in the liver. In T2 mice the Neomycin gene has been 13 inserted in the first intron of the PBGD gene. Homozygous condition is lethal and heterozygotes 14 animals exhibit a 43% loss of PBGD activity in the liver. Neither of the strains showed signs of porphyria nor increased urinary excretion of heme precursors after treatment with phenobarbital 16 (Pb) and/or estradiol (data not shown). To further lower the PBGD activity, crossbreeding the 17 two strains was performed. Compound heterozygotes animals, carrying knockout alleles for both 18 Ti and T2 are used as a disease model for AIP. These mice exhibit the typical biochemical 19 characteristics of human porphyria, notably, decreased hepatic PBGD
activity and massively increased urinary excretion of heme precursors after treatment with drugs such as phenobarbital.
21 Porphyrins, mostly uroporphyrin and coproporphyrin, are also elevated in AIP but increased 22 urinary porphyrin is a much less specific feature than increases in PBG and ALA levels.
23 Behavioural tests such as the rotarod test reveal decreased motor function after Pb administration 24 and histopathological findings include axonal neuropathy and decreased nerve conduction with aging.
26 1.1.2 AAV vector 27 Plasmids and sequences 28 The AAV plasmids used in this study contain an expression cassette flanked by two ITRs 29 from the AAV2 and an appropriate stuffer sequence to adjust the size of the AAV genome to the optimal packaging capacity described for AAV. The transgene expression cassette has the 31 following elements: the 5'ITR from AAV2, a liver-specific promoter Ea1bAATp with regulatory 22094617.1 23 Agent Ref: 77629/00002 1 sequences from the albumin enhancer (Kramer et al., 2003, Mol Ther. 7(3):375-85), the 2 housekeeping PBGD cDNA (GenBank acc # X04808) or Luciferase reporter gene (GenBank acc 3 # MI5077). The bovine growth hormone polyadenylation sequence [bGH poly(A)]
(bases 2326-4 2533 GenBank acc # M57764), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (bases 1021-1750 GenBank acc # J04514) was added to enhance transcription 6 (Donello et al., 1998 J Virol. 72(6):5085-92) and the 3'ITR from AAV2. These two AAV
7 plasmids were named ssAAV-polyA-EalbAAT-PBGD-WPRE (expressing the therapeutic gene) 8 and ssAAV-polyA-EalbAAT-Luciferase-WPRE (expressing the reporter gene GFP).

Preparation of AA V vectors 11 AAV2/8 vectors were produced by calcium phosphate-mediated co-transfection in 293 12 cells of three different plasmids pAdDeltaF6, p5E18-VD2/8 and the therapeutic (AAV-polyA-13 EalbAAT-PBGD-WPRE) or reporter gene (AAV-polyA-EaIbAAT-Luciferase-WPRE), 14 (Hermens et al, 1999 Hum Gene Ther. 10(11):1885-91; and Gao et al 2002, Proc Natl Acad Sci USA, 99(18):11854-9). Briefly, 293 cells were co-transfected with pAdDeltaF6, p5E18-VD2/8 16 and target vector by calcium phosphate and the virus was harvested by freeze-thawing of the 17 cells, 48h after transfection. The virus was purified by ion exchange column chromatography and 18 iodixanol gradient centrifugation followed by filtration and further concentration against 19 phosphate-buffered saline (PBS)-5% sucrose. Virus titres in terms of genome copies/ml were determined by Q-PCR performed in triplicate, TaqMan (AppliedBiosystems) analysis using 21 primers pr300fw 5'CCCTGTTTGCTCCTCCGATAA3' pr30lrv 5' 22 GTCCGTATTTAAGCAGTGGATCCA 3' amplifying a 95 bp fragment of the hAAT
promoter 23 region. Protein composition and purity was determined by sodium dodecyl sulfate-24 polyacrylamide gel electrophoresis (SDS-PAGE).
1.1.3 Experiment Set-Up 26 Preliminary test 27 In order to test the infective capability and to evaluate the PBGD
expression of the 28 AAV2/8 vector, two AIP animals were injected with a dose of 9x1012 vg of AAV2/8-PBGD and 29 sacrificed at day 6.

22094617.1 24 Agent Ref: 77629/00002 2 Proof-of-principle assay 3 We evaluated the AAV2/8-mediated liver transduction by comparing levels of PBGD
4 expression in the livers of AIP mice after injection of AAV2/8-hBGD.
Compound heterozygous AIP mice in C57B1/6 background of 12 to 25 weeks age (10 mice per group 5 males and 5 6 females) were injected intravenously, via the tail vein, with a total of 100-200 l.tl corresponding 7 to 5e12 vg/kg of AAV2/8-hPBGD or AAV2/8-luciferase control vector. Two additional groups 8 of five AIP male mice (of the same age that the previous ones) were injected with 5el l vg/kg or 9 5e10 vg/kg of AAV2/8-hPBGD. Extra group of wild type and AIP mice will be included. Dose scheme is shown in Table 2.

12 Table 2. Vector doses (Gcs) used as determined by Q-PCR

Gs/ml Gcs/mice sex n vector PBGD 1.73E12 1E11 a' 5 1E10 a' 5 1E9 a' 5 Luciferase 1.79E12 1E11 a' 3 At fifteen, twenty-eight and ninety days after rAAV vector injection, motor disturbance 16 and porphyrin precursor accumulation was measured in mice before and after the acute attack 17 induced by phenobarbital injection. For this, phenobarbital (Pb, diluted in saline) was 18 administered intraperitoneally once a day on 4 consecutive days with increasing doses (75, 80, 19 85, 90 mg/kg body weight).
Nerve conduction measurement 21 Electrophysiological studies were carried out to demonstrate axonal degeneration and loss 22 of myelin in sciatic nerve. Two male AIP mice of 6 months old were injected with 1e12 gc/kg.
23 Animals were treated biweekly with repeated doses of Pb to accelerate the motor neuropathy.
22094617.1 25 Agent Ref: 77629/00002 1 Nerve conduction measurements were performed in animals at 11 and 14 months old, before and 2 after the acute attack induced by Pb.
3 1.2. Results 4 1.2.1 Proof-of-principle assay Effects of treatment on heme precursors levels throughout the study 6 Fifteen, twenty-eight and ninety days after virus injection, AIP mice were treated with 7 increasing dose of Pb for 4 days and the levels of heme precursors were measured in urine.

8 As expected, male AIP mice from the control group (AAV-Luc, Figure 1) displayed 9 increased excretion of precursors after Pb injection, whereas no variation in the excretion of precursors occurred in mice injected with high doses of AAV2/8-PBGD (Figure 1). Complete 11 avoidance of ALA and PBG accumulation after phenobarbital-induction began at 5eI Ivg/kg in 12 male AIP mice (Figure 1). Partial prevention was observed in AIP males treated with 5e10 vg 13 AAV-PBGD/kg. There were no changes in the profile of phenobarbital-induced ALA and PBG
14 excretion two weeks after the AAV administration and at the end of the study (Figure 1).
The administration of therapeutic vector (5e12 vg of AAV2/8-PBGD/kg) in female animals 16 also prevented Pb-induced acute attacks, as shown by the lack of abnormal accumulation of 17 porphyrin precursors in urine (Figure 2). In the control group (injected with AAV-Luc), female 18 AIP mice (Figure 2) exhibited less porphyrin precursor accumulation than male AIP animals 19 (Figure 1) due to a lower hepatic activity of the rate-limiting ALAS I
enzyme in females when compared to male AIP mice (data not shown).
21 Effects of AAV administration on motor disturbance throughout the study:
Rotorod test 22 As expected, AIP mice exhibit motor disturbance when compared with wild type animals. Motor 23 disturbance in AIP mice treated with control vector (AAV-Luc) was exacerbated after 24 phenobarbital administration both in male and female (data not shown). The Pb-induced motor disturbance in male AIP mice was almost completely abolished in animals treated with the 26 therapeutic vector both at the beginning (fifteen and twenty-eight days after the AAV injection, 27 data not shown) and at the end of the study. Male and female AIP animals treated ssAAV2/8-28 hPBGD showed complete protection against the Pb-induced motor disturbance during the whole 29 period of the study (fifteen and twenty-eight days after the AAV injection, and at the end of the study (Figure 13). In males, the mean length in time that a mouse that received the luciferase 31 vector could stay on the dowel was approximately 200 seconds. However, upon porphyric attack, 22094617.1 26 Agent Ref: 77629/00002 1 this was halved to --100 seconds. In the males that received all three different doses of PBGD
2 vector, mice could stay on the dowel for nearly as long as when not experiencing an attack, 3 although not to unchallenged wild type levels. The same was also found in females.

Effects of the ssAA V-hPBGD administration on liver PBGD level at sacrifice:
Western blot 6 analysis.
7 As expected, the administration of the control vector (ssAAV-Luc) did not increased 8 PBGD expression both in males (Figure 3) and female mice (Figure 4). In males, dose-dependant 9 increase of hepatic PBGD expression was observed by western blot analysis (Figure 3). Different patterns of migration between endogenous and human PBGD allows for identification of 11 exogenous PBGD (Figure 3).
12 In females, high expression of PBGD was observed in the liver of mice treated with 5e12 13 vg/kg of ssAAV-hPBGD (Figure 4).
14 Effects of the AA V-PBGD administration on liver PBGD activity at sacrifice The measurement of enzymatic activity confirms that the PBGD protein expressed by the 16 therapeutic vector is functionally active (Figures 5 and 6). No significant differences in the 17 PBGD activity (p = 0.35) was observed between males (Figure 5) and females (Figure 6) injected 18 with higher doses of the therapeutic vector (AAV-PBGD). In males, a dose-dependant increase 19 of liver PBGD activity was observed (Figure 5). Mice administered with low doses of therapeutic vector (5e10 vg AAV-PBGD /kg) show the same PBGD activity as wild type animals 21 Immunohistochemistry analysis: distribution of the PBGD expression in the liver 22 Immunohistochemistry was used to determine PBGD protein levels in individual cells and 23 was performed on duplicate slides for each treatment group. For PBGD
immunohistochemical 24 staining a polyclonal antibody anti-PBGD was developed in LIMA. The antibody antiPBGD
recognizes the endogenous protein and the exogenous human protein mediated by the 26 ssAAV2/8-hPBGD (data not shown).
27 The liver of AIP animals injected with the control virus show low expression of PBGD in 28 the cytoplasm of whole hepatocytes as detected by immunohistochemistry analysis (data not 29 shown). This weak signal in the cytoplasm corresponds to the endogenous PBGD. Cell nuclei were counterstained by hematoxylin to give a blue background contrast to brown colour of the 31 positive reaction (data not shown).

22094617.1 27 Agent Ref: 77629/00002 1 In male AIP mice injected with therapeutic vector, a dose-dependant increase of PBGD
2 expression was observed. High PBGD expression was observed in male mice injected with the 3 highest doses of therapeutic virus. The area of strong PBGD expression was reduced in the 4 animals injected with intermediate dose of therapeutic virus and it was reduced to isolated cells in the animals injected with low doses of virus. These results correlate well with those obtained 6 previously by western and enzymatic activity in the same animals.
7 No significant differences in the PBGD immunohistochemical staining was observed 8 between males and females injected with the same dose of the therapeutic vector (5e12 vg AAV-9 PBGD/kg). Males and females receiving higher doses of therapeutic vector exhibited a high expression of PBGD in both parenchyma and around the vessels (data not shown).
11 PBGD was reported to be localized in the cytoplasm of the cells, however it has previously 12 reported that PBGD is imported into the nucleus of various cell lines and of primary cells 13 (Grunberg-Etkovitz et al. 2006, Biochim Biophys Acta.1762(9):819-27). In our mice injected 14 with the control virus, most of the nucleus exhibit a characteristic blue stain (due to hematoxylin) and had few nuclei were immunostained brown (due to DAB; data not shown).
However, in our 16 mice injected with AAV-PBGD we observed a high proportion of brown, reflecting a high 17 nuclear distribution of PBGD protein. The proportion of high PBGD positive nuclei was 18 measured in different groups. Again, a dose-dependent increase of hepatocytes expressing high 19 nuclear PBGD protein was observed in males.
Neurological evaluation of peripheral neuropathy 21 In another aspect, we have performed a neurological evaluation of peripheral neuropathy in 22 AIP mice that included histological analysis of sciatic nerves (data not shown) and functional 23 studies of motor potential evoked by proximal stimulation of the sciatic nerve (data not shown).
24 Normal axon density observed in AIP animals transduced with rAAV2/8-PBGD
vector suggested that hPBGD over-expression in the liver facilitated axon regeneration of the sciatic 26 nerve in AIP mice (Figure 12).
27 Electrophysiological studies were carried out to demonstrate axonal degeneration and loss 28 of myelin in sciatic nerve. This shows that long term expression of the PBGD transgene protects 29 against functional block induced by phenobarbital administration. Motor potential evoked by proximal stimulation of the sciatic nerve in wild-type, young AIP, old AIP and AIP mice 31 transduced with rAAV2/8-PBGD before and after seven acute attacks of porphyria induced by 22094617.1 28 Agent Ref: 77629/00002 1 phenobarbital shows restoration of function in terms of latency, duration and amplitude (data not 2 shown).

4 Example 2: Increased in vivo enzymatic activity from codon-optimized PBGD
cDNA
2.1 Construction of the plasmids used for hydrodynamic injection and AAV gene delivery with 6 wtPBGD and coPBGD
7 Plasmids used in this study contain expression cassettes with the following elements in a 5' 8 to 3' order: a liver-specific promoter EalbAATp with regulatory sequences from the albumin 9 enhancer (Kramer et al., 2003, Mot Ther. 7(3):375-85), the housekeeping (i.e. non-Erythroid) PBGD cDNA (GenBank acc # X04808). The 3' UTR from the PBGD (bases 1463-1487 11 GenBank acc #NM_000190) and the PBGD polyadenylation sequence [poly(A) PBGD] (bases 12 9586-9629 GenBank acc # M95623). The two plasmids containing these expression cassettes are 13 named psi1180-pAAT-PBGD-PolyA PBGD and psi1180-pAAT-coPBGD-PolyA PBGD and 14 differ only in the PBGD coding sequence as explained below.
To generate a codon-optimized PBGD, the gene Bank sequence # NM_000190 16 corresponding to the human PBGD non-erythroid cDNA was adapted in codon usage to the bias 17 of Homo sapiens (Codon Adaptation Index value 0.97). Further modifications in the coPBGD
18 coding sequence included: 1) A Kozak sequence was introduced to increase translational 19 initiation; 2) Two stop codons were added to ensure efficient termination;
and 3) CG content was increased from 55% to 65%. The final coPBGD cDNA, after optimization, has 195 bp changes 21 and 82.1% homology with the original cDNA sequence # NM_000190. This sequence (SEQ ID
22 NO: 1) was synthesized and subcloned in plasmid psil180-pAAT-PBGD-PolyA
PBGD
23 replacing the sequence of the WT PBGD cDNA. The new generated plasmid was named 24 psll 180-pAAT-coPBGD-PolyA PBGD.
The entire expression cassette sequence from plasmids psi 1180-pAAT-PBGD-PolyA
26 PBGD and ps11180-pAAT-coPBGD-PolyA PBGD were subcloned in plasmid pVD155, a 27 plasmid containing the 2 ITRs from AAV2. The resulting plasmids named pVD
153 and 28 pVD 191 where cotransfected each of them with a parental baculovirus genome into SF9 cells to 29 generate the recombinant baculovirus Bac.VD153 and Bac.VD191. These baculoviruses were used to generate AAV5 vectors in insect cells. Briefly, SF9+ cells were co-infected with 3 31 different recombinant baculoviruses: Bac.VD92, Bac.VD88 and Bac.VD153 (baculovirus 22094617.1 29 Agent Ref: 77629/00002 1 containing the therapeutic EalbAAT-PBGD-polyA). For the production of an AAV5 vector 2 containing the expression cassette EalbAAT-coPBGD-polyA the baculovirus Bac.VD191 was 3 used instead of Bac.VD153.
4 The AAV5 vector was harvested by freeze-thawing of the cells, 72 h after infection. The vector was purified by affinity column chromatography followed by filtration and further 6 concentration. Virus titres in terms of genome copies/ml were determined by TaqMan Q-PCR, 7 (AppliedBiosystems) analysis using primer hAAT taq reverse 8 5'CAGCGTCCTGTGTCCAAGGT3', primer hAAT taq forward 9 5'AGGCCAACTTGTCTACGTTTAGTATG3' (both from MWG-Biotech AG)and probe hAAT
5'CTGTAGATCTGTACCCGCCACCCCC3' (MWG-Biotech AG). Protein composition and 11 purity was determined by SDS-PAGE.
12 2.2 Enzymatic PBGD assays 13 PBGD activity in tissue homogenates was determined by measuring the conversion of PBG
14 to uroporphyrin according to the method of Anderson and Desnick (Anderson PM, Desnick RJ., 1982, Enzyme, 28(2-3):146-57). Briefly, 1 g of tissue was homogenized at 4 C
in 4 volumes of a 16 1.15% KCl solution. Homogenate was centrifuged at 12.000 rpm at 4 C for 20 minutes and the 17 clear supernatant without any cellular debris was used the same day for protein determination 18 (Bradford using an albumin standard) and PBGD activity.
19 The supernatant samples were diluted 1:3 with phosphate buffer (pH 7.6), DTT, C12Mg and Triton X-100; and 100 l of this mixture was preincubated with 1.8 ml of Tris-HC1 O.1M (pH
21 8.1) for 3 min at 37 C. Next, the mixture was incubated in the dark with 0.5 ml 1 mM PBG
22 substrate for 60 minutes at 37 C. The reaction was stopped with 350 gl cold TCA 40% and the 23 uroporphyrinogen formed was oxidised to uroporphyrin after light exposure.
Uroporphyrins were 24 measured quantitatively using a spectrofluorometer with an excitation peak (?, ext) at 405 nm and window emission peak (), em) values between 550-660 nm. The PBGD activity was expressed in 26 terms of pmol uroporphyrin/mg protein/hour using appropriate standards.
27 2.3 Hydrodynamic injection of PBGD and coPBGD plasmids 28 50 g of each plasmid ps11180-pAAT-PBGD-PolyA PBGD or ps11180-pAAT-coPBGD-29 PolyA PBGD dissolved in 2.5 ml PBS were injected hydrodynamically in the lateral tail vein of AIP mice (n=4) to deliver the plasmid to the liver. Mice were sacrificed 1 week after the 31 injection and PBGD enzymatic activity in the liver and kidney homogenates was determined.

22094617.1 30 Agent Ref: 77629/00002 1 coPBGD enzyme expressed by hepatocytes after hydrodynamic delivery of the psi 1180-2 pAAT-coPBGD-PolyA PBGD plasmid resulted in 30% more active PBGD in liver homogenates 3 compared to mice that received the wild type (wtPBGD) psl I 180-pAAT-PBGD-PolyA PBGD
4 plasmid. The values of the PBGD activity expressed as pmol uroporphyrin/mg protein/hour were 5.54 1.64 and 7.30 0.913 for wtPBGD and coPBGD respectively (p=0.0317, two-tailed 6 Mann Whitney test). The levels of vector DNA present in the liver was confirmed by Q-PCR
7 using primers that hybridize to the transgene. A ratio between the DNA
copies of the endogenous 8 housekeeping gene GADPH and the DNA copies of the PBGD transgene in each mouse was 9 calculated. DNA ratios (2.950 2.25 and 1.760 0.6804 for PBGD and coPBGD
respectively) did not show any significative difference and demonstrated that approximately the same number 11 of copies of the therapeutic vector had been delivered to the liver by hydrodynamic injection.
12 Data are represented in Figure 7.
13 2.4 Proof-of-principle for PBGD gene therapy with a PBGD-AAV2/5 vector in AIP mice 14 AIP mice described before in section I.I.I. were used to prove the therapeutic effect of AAV2/5-EalbAAT-PBGD-polyA.
16 A dose of 5e12 gc/kg of AAV2/5-EalbAAT-PBGD was intravenously injected in AIP mice 17 (males and females). Control animals received the same vector but carrying the luciferase 18 reporter gene. Two, four and thirteen weeks after the AAV2/5-EalbAAT-PBGD
administration, 19 animals were treated with increasing doses of Pb for 4 days to induce the porphyric attack.
Twenty four hours after the last dose of Pb, the levels of heme precursors were measured in 24 21 hours-urine samples and motor co-ordination was analysed using the rotarod test. Mice were 22 sacrificed three months after the AAV administration to quantify the amount of PBGD produced 23 in the liver.
24 The basal levels of ALA and PBG in AIP mice were: 88 24 and 16 5 gg/mg creatinine in males and 87 19 and 14 8 gg/mg creatinine in females. A dose of 5e12 gc/kg was able to 26 prevent the effect of Pb in the precursors ALA and PBG in both males (118 34 and 11 4 g/mg 27 creatinine) and females (52 5 and 51 24 gg/mg creatinine) as is shown in Figure 8 (males) and 28 Figure 9 (females). Animals treated with the same dose of an AAV2/5-EalbAAT-Luciferase 29 showed a high excretion of ALA and PBG precursors after Pb injection (1418 659 and 1184 585 gg/mg creatinine in males and 295 91 and 298 181 g/mg creatinine in females).

22094617.1 31 Agent Ref: 77629/00002 1 Motor disturbance induced by Pb treatment in AIP mice was almost completely abolished 2 in animals treated with the therapeutic vector, as measured in the Rotarod test (data not shown).
3 Three months after the AAV administration, all animals were sacrificed and enzymatic 4 PBGD activity measured from livers of all animals (Figure 10). Males injected with a dose of 5e12 gc/kg of AAV5-EalbAAT-PBGD expressed 26.1 7.3 pmol URO/mg protein/h of PBGD
6 in the liver. This amount represents an over-expression of 10 times when compared with AIP
7 mice receiving reporter gene Luc (2.4 0.4 pmol URO/mg protein/h) and 3 times the levels of a 8 wild type mice (7.9 1.6 pmol URO/mg protein/h). A dose of 5e12 gc/kg did reveal differences in 9 the liver PBGD enzymatic activity in females (18.5 6.3 pmol URO/mg protein/h).
2.5 AAV5-mediated liver-specific expression of PBGD and coPBGD in AIP mice 11 We evaluated the AAV2/5-mediated liver transduction by comparing levels of PBGD
12 expression in the livers of AIP mice after injection of AAV2/5-PBGD or AAV2/5-coPBGD.
13 Male AIP mice in C57B1/6 background of 12 to 25 weeks old were injected intravenously with a 14 total of 200 tl corresponding to 1.25e11 vg of AAV2/5-PBGD (n=22) or AAV2/5-coPBGD
(n=24). At 1, 2 and 3 months after virus injection, mice were sacrificed and livers were collected 16 to determine the enzymatic activity of PBGD in liver homogenates. Results were expressed as 17 mean SEM of the PBGD and comparison between means was performed using the Mann 18 Whitney test (Figure 11 A).
19 In accordance with Lindberg et al., Nature Genetics 12: 195-199, 1996, AIP
mice expressed only 30% of the PBGD enzymatic activity in the liver when compared with WT mice (2.53 0.15 vs 21 7.94 0.94 pmol URO/mg protein/h, respectively). Besides, the levels of PBGD
expression after 22 AAV2/5-PBGD and AAV2/5-coPBGD gene transfer were statistically different at all points after 23 administration. 64.00 5.99 vs 86.23 6.82 for AAV2/5-PBGD and AAV2/5-coPBGD
24 respectively after 1 month of infection (p=0.019) and 78.98 9.69 vs 111.50 10.20 AAV2/5-PBGD and AAV2/5-coPBGD respectively after 2 months of infection (p=0.038).
Values of 26 enzymatic activity in the murine livers are shown in Figure 11.A.
27 Semiquantitative PCR analysis of AAV vector genome was performed. The levels at 1, 2 28 and 3 months post-injection were determined and are shown in Figure 11.B.
and Q-PCR data 29 corroborate that the amount of viral plasmid was similar in both cohorts of animals The immunohistochemical analysis of the liver with PBGD specific antibodies revealed 17% and 31 21% of PBGD expressing cells in mice infected with AAV2/5-PBGD and AAV2/5-coPBGD
22094617.1 32 Agent Ref: 77629/00002 1 respectively (Figure 11 .C and D) representing essentially similar transduction efficiencies, which 2 were maintained for the 3 months period of study 22094617.1 33

Claims (21)

1. A nucleic acid comprising a nucleotide sequence coding for a human porphobilinogen deaminase, wherein at least 320 of the codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID NO: 1 or wherein at least 305 of the codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID NO: 3.

2. A nucleic acid according to claim 1, wherein the nucleotide sequence coding for the human porphobilinogen deaminase has at least 95% identity over the entire length of SEQ ID NO: 1 or

3, as determined by a Needleman and Wunsch global alignment algorithm.

3. A nucleic acid according to claim 1 or 2, wherein the nucleotide sequence codes for the amino acid sequence of SEQ ID NO: 2 or 4.

4. A nucleic acid according to claim 3, wherein the nucleotide sequence has the nucleotide sequence of SEQ ID NO: 1 or 3.

5. A nucleic acid construct comprising a nucleotide sequence coding for a human porphobilinogen deaminase, wherein at least 320 of the codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID NO: 1 or wherein at least 305 of the codons coding for the human porphobilinogen deaminase are identical to the codons in SEQ ID NO: 3.

6. A nucleic acid construct according to claim 5, wherein the nucleotide sequence coding for the human porphobilinogen deaminase has at least 95% identity over the entire length of SEQ ID
NO: 1 or 3, as determined by a Needleman and Wunsch global alignment algorithm.

7. A nucleic acid construct according to claim 5 or 6, wherein the nucleotide sequence codes for the amino acid sequence of SEQ ID NO: 2 or 4.

8. A nucleic acid construct according to claim 7, wherein the nucleotide sequence has the nucleotide sequence of SEQ ID NO: 1 or 3.

9. A nucleic acid construct according to any one of claims 5 to 8, wherein the nucleotide sequence coding for the human porphobilinogen deaminase is operably linked to a promoter for expression in human cells.

10. A nucleic acid construct according to claim 9, wherein the promoter is a liver-specific promoter.

11. A nucleic acid construct according to claim 10, wherein the liver-specific promoter is selected from the group consisting of an .alpha.1-anti-trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a thyroxin-binding globulin (TBG) promoter, an Hepatic Control Region (HCR)-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an AAT promoter combined with the mouse albumin gene enhancer (Ealb) element and an apolipoprotein E promoter

12. A nucleic acid construct according to claim 11, wherein the promoter has the sequence of SEQ ID NO: 5.

13. A nucleic acid construct according to any one of claims 1 to 8, wherein the construct is in the form of a viral gene therapy vector, preferably a parvoviral vector.

14. A parvoviral virion comprising a nucleic acid as defined in any one of claims 1 to 4 or a nucleic acid construct as defined in any one of claims 5 to 13.

15. A parvoviral virion as defined in claim 14 for use as a medicament or for use in the treatment of the human or animal body by therapy.

16. A parvoviral virion as defined in claim 14 or 15 for use in the treatment of a condition caused by a deficiency in porphobilinogen deaminase.

17. A parvoviral virion according to claim 16, wherein the condition is acute intermittent porphyria.

18. A pharmaceutical composition comprising a parvoviral virion as defined in claim 14 and a pharmaceutically acceptable carrier.

19. Use of a parvoviral virion as defined in claim 14 for use in the manufacture of a medicament for use in the treatment of a condition caused by a deficiency in porphobilinogen deaminase.

20. A method for the delivery of a nucleotide sequence encoding porphobilinogen deaminase to a mammal which method comprises:
a. providing a parvoviral virion as defined in claim 14 or a pharmaceutical composition as defined in claim 18; and b. administering said parvoviral virion to a mammal under conditions that result in the expression of protein at a level that provides a therapeutic effect in said mammal.

21. A method for treating a condition caused by a deficiency in porphobilinogen deaminase wherein the method comprises the step of administering an effective amount of a parvoviral virion as defined in claim 14 or a pharmaceutical composition as defined in claim 18 to a subject with a porphobilinogen deaminase deficiency, wherein preferably the condition is acute intermittent porphyria.